Toxic Effect Of Copper Exposure On Germ Cells Of Male Mice

Copper,as an essential trace element in the organism,is an important part of various copper enzymes.However,copper is also one of the most serious sources of environmental pollution.Copper pollution is the most serious problem,especially in the vicinity of metal smelters and copper mining areas.There have been many reports of the toxic effects of copper in the reproductive system and other organs and tissues that have threatened the health of humans,animals and plants.However,there have been no systematic studies on the effects of copper exposure on male reproductive toxicity.Hence,this study aims to explore the effects of copper on testicular tissue and GC-1 spermatogonia in both in vivo and in vitro.And we initially explored the mechanism of copper toxicity in spermatogenic cell damage to systematically evaluat the toxic effect of copper exposure on male reproduction.In vivo: In this study,a copper exposure model was established by intragastric administration of anhydrous copper sulfate.Sixty CD-1 adult male mice were randomly and equally divided into four groups: control group(Ctr,ultrapure water),and test group 1(Tes1,25 mg/kg/d),test group 2(Tes2,100 mg/kg/d)and test group 3(Tes3,150 mg/kg/d)were continuously administered for 8 weeks.The correlation between serum copper and testis copper was analyzed by measuring the copper content in serum and testis.Perform sperm counts and analyze sperm motility.Observe the pathological changes of the testis and analyze the degree of copper damage to the testis.The activity of testosterone and testosterone synthesis related enzymes in serum and/or testis was measured to evaluate the effect of copper on testosterone synthesis.The mRNA expression levels of testosterone synthesis-related genes and apoptosis-related genes were examined to evaluate the effect of copper on testosterone synthesis and testicular cell apoptosis from the gene level.The expression level of Casp3 protein in the testis tissues was detected to evaluate the effect of copper on apoptosis from the protein level.The results showed that serum copper levels increased significantly with increasing doses of copper in the stomach(P<0.01).The copper content in the testis tissue was significantly higher in the Tes3 group(P<0.05).The covariance between the two groups of data and their correlation were analyzed.The coefficients were 2.73 and 0.81,respectively,suggesting a strong correlation between serum copper and testicular copper.Both sperm count and sperm motility decreased significantly with increasing copper dose(P<0.05).Compared with the Ctr group,the testicular tissue of the higher copper dose group was severely damaged,mainly due to the reduction of spermatogonia and spermatocytes,cavity in the seminiferous tubule tissue,and even the phenomenon of connective tissue filling.The testosterone content in serum and testis,the expression of testosterone synthesis related enzymes in the testis,and the expression of testosterone synthesis genes were not significantly different from those in the Ctr group(P>0.05).Higher concentration of copper in the testis significantly increased the mRNA expression of apoptosis genes Bax and Casp3 and decreased the expression of anti-apoptosis gene Bcl2(P<0.05).High copper significantly increased the expression of Casp3 protein in mouse testicular cells(P<0.05).In vitro: In order to study the effect of copper exposure on the cytotoxicity of male mouse GC-1 spermatogonia,spermatogonia were treated with culture medium containing copper at final concentrations of 10,50,and 100 ?M,and incubated for 24 h before detection of intracellular changes in ROS,MDA,T-AOC,CAT,GSH,ATP levels,changes in mitochondrial membrane potential,LDH content in supernatant of cell culture supernatants,and immunofluorescence staining for early/late apoptotic status of spermatogonia(P<0.05).Fluorescence quantitative PCR was used to detect the mRNA expression levels of apoptosis and autophagy-related genes.The results showed that compared with Ctr group,the levels of ROS,MDA,and T-AOC in spermatogonia treated with higher copper concentrations increased to varying degrees,and the LDH in the supernatant of cell culture supernatant of each test group was increased(P<0.05).The contents of CAT and GSH increased,but the contents of CAT and GSH increased first and then decreased(P<0.05).The mitochondrial membrane potential and ATP levels showed a significant decrease(P<0.05).The number of apoptotic cells and dead cells increased with the copper dose(P<0.05).Higher concentrations of copper significantly increased mRNA expression levels of apoptosis genes Bax,Casp8,Casp3 and autophagy genes Atg3,Atg5,p62,and Lc3 b and decreased the expression of anti-apoptosis gene Bcl2(P<0.05).Conclusions: It can be concluded from this study that high copper induces damage to testicular tissue and affects the process of spermatogenesis.The potential mechanism of this effect may be that high copper induces oxidative stress in spermatogonia,leading to apoptosis and autophagy.